Nitric Oxide (NO) is a gas that has multiple signalling and effector functions in mammalian tissues. Hemoglobin scavanges NO and this limits NO's biological activity; conversely, it has recently been proposed that reversible NO binding to the beta- globin cysteine 93 is allosterically linked to oxygenation and this contributes to the control of oxygen delivery in tissues. We have investigated NO transport and metabolism by hemoglobin in human subjects in vitro and in vivo. We have developed new, highly sensitive cNitric Oxide (NO) is a gas that has multiple signalling and effector functions in mammalian tissues. Hemoglobin scavanges NO and this limits NO's biological activity; conversely, it has recently been proposed that reversible NO binding to the beta- globin cysteine 93 is allosterically linked to oxygenation and this contributes to the control of oxygen delivery in tissues. We have investigated NO transport and metabolism by hemoglobin in human subjects in vitro and in vivo. We have developed new, highly sensitive chemical and spectrophotometric assays for S- nitrosohemoglobin (SNO-Hb) and nitrosyl (heme) hemoglobin (Hb-Fe II-NO). We find that inhalation of NO at 80 ppm by normal subjects leads to formation of significant amounts of nitrosylated hemoglobin with a large arteriovenous gradient, largely due to formation of methemoglobin >> Hb-Fe II-NO SNO- Hb. The primary cause of the A-V difference is the Hb-FE II- NO. In addition, we find an A-V gradient in red cell nitrate and nitrate levels. These results confirm the existence of the SNO-Hb pathway but suggest that reversible binding to ferrous heme is a more important mechanism. Similar results have been obtained with sickle cell patients; in vitro red cell studies with NO show the dominance of methemoglobin formation. Our current data suggests that bioactive NO is transported by either the nitrosyl(heme) hemoglobin or the nitrite and we have studies in progress to distnguish these mechanisms. We are also using a variety of physiological measures of blood flow- including MRI and reflectance spectroscopy - to examine the effects of NO inhalation on regional blood flow and have found that after inhibition of local NO synthesis, inhaled NO causes peripheral effects in normal individuals but not in sickle cell patents. Recently we have shown that this is due to the presence of circulating hemoglobin in the plasma of the sickle cell patients. In particular, we have identified the biologically active species as the hemoglobin dimer-haptoglobin complex. We believe that this is an important factor in contributing to the pathophysiology of this disease, as well as in thalassemia and other chronic anemias. Certain new therapeutic approaches are also opened if this mechanism is indeed has a major role in determining the metabolism of NO.